type i interferon signals in macrophages and dendritic...
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Type I Interferon Signals in Macrophages and Dendritic Cells ControlDengue Virus Infection: Implications for a New Mouse Model To TestDengue Vaccines
Roland Züst,a Ying-Xiu Toh,a Iris Valdés,b Daniela Cerny,a Julia Heinrich,c Lisset Hermida,b Ernesto Marcos,b Gerardo Guillén,b
Ulrich Kalinke,c Pei-Yong Shi,d,e Katja Finka
Singapore Immunology Network, Agency for Science, Technology and Research, Singaporea; Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cubab;
Institute for Experimental Infection Research, Twincore, Center for Experimental and Clinical Infection Research, Hannover, Germanyc; Novartis Institute for Tropical
Diseases, Chromos, Singapored; Wadsworth Center, New York Department of Health, Albany, New York, USAe
ABSTRACT
Dengue virus (DENV) infects an estimated 400 million people every year, causing prolonged morbidity and sometimes mortal-ity. Development of an effective vaccine has been hampered by the lack of appropriate small animal models; mice are naturallynot susceptible to DENV and only become infected if highly immunocompromised. Mouse models lacking both type I and typeII interferon (IFN) receptors (AG129 mice) or the type I IFN receptor (IFNAR�/� mice) are susceptible to infection with mouse-adapted DENV strains but are severely impaired in mounting functional immune responses to the virus and thus are of limiteduse for study. Here we used conditional deletion of the type I IFN receptor (IFNAR) on individual immune cell subtypes to gen-erate a minimally manipulated mouse model that is susceptible to DENV while retaining global immune competence. Mice lack-ing IFNAR expression on CD11c� dendritic cells and LysM� macrophages succumbed completely to DENV infection, while micedeficient in the receptor on either CD11c� or LysM� cells were susceptible to infection but often resolved viremia and recoveredfully from infection. Conditional IFNAR mice responded with a swift and strong CD8� T-cell response to viral infection, com-pared to a weak response in IFNAR�/� mice. Furthermore, mice lacking IFNAR on either CD11c� or LysM� cells were also suffi-ciently immunocompetent to raise a protective immune response to a candidate subunit vaccine against DENV-2. These datademonstrate that mice with conditional deficiencies in expression of the IFNAR represent improved models for the study ofDENV immunology and screening of vaccine candidates.
IMPORTANCE
Dengue virus infects 400 million people every year worldwide, causing 100 million clinically apparent infections, which canbe fatal if untreated. Despite many years of research, there are no effective vaccine and no antiviral treatment available fordengue. Development of vaccines has been hampered in particular by the lack of a suitable small animal model. Mousemodels used to test dengue vaccine are deficient in interferon (IFN) type I signaling and severely immunocompromisedand therefore likely not ideal for the testing of vaccines. In this study, we explored alternative models lacking the IFN re-ceptor only on certain cell types. We show that mice lacking the IFN receptor on either CD11c- or LysM-expressing cells(conditional IFNAR mice) are susceptible to dengue virus infection. Importantly, we demonstrate that conditional IFNreceptor knockout mice generate a better immune response to live virus and a candidate dengue vaccine compared toIFNAR mice and are resistant to subsequent challenge.
Dengue virus (DENV, a member of the Flaviviridae family, isa mosquito-borne pathogen that infects approximately 400
million people every year (1, 2). Each of the four DENV serotypescauses a spectrum of clinical symptoms ranging from mild fever topotentially fatal manifestations of dengue shock syndrome.DENV causes an acute infection with high fever, which usuallyresolves after 5 to 7 days. At this time, most patients have clearedthe high virus load. Intriguingly, however, this is also the timepoint when some patients start to develop vascular leakage, which,if untreated, can lead to a collapse of the metabolism and organfailure. The frequency, severity, and geographical spread of caseshas increased over the past decades (3, 4), and DENV infection isnow considered a leading cause of morbidity in the tropics.
There are no effective treatments for dengue fever, and thedevelopment of a vaccine has been hampered by the lack of suit-able small animal models. Wild-type (wt) mice are not susceptibleto infection with field strains of DENV, and while viral replication
in these animals can be forced by intracranial injections of high-titer mouse-adapted DENV strains, the resulting clinical diseasebears little resemblance to dengue fever in humans. Humanizedmice, which are engrafted with human progenitor cells, provide asystem to study human T-cell responses in vivo. However, it hasbeen difficult to generate a sustained antibody production (re-viewed in reference 5). Nonhuman primates (NHPs) have beenused to test candidate dengue vaccines but do not show clinical
Received 26 December 2013 Accepted 7 April 2014
Published ahead of print 16 April 2014
Editor: A. García-Sastre
Address correspondence to Roland Züst, [email protected].
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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symptoms upon challenge. In addition, the ethical issues, biocon-tainment requirements, and high cost of using NHPs serve tohighlight the urgent need for a rodent model.
Interferons (IFNs) are critical mediators of protection againstviral infection, and mice lacking the receptors for both alpha/betaIFN (IFN-�/�) (type I IFNs) and IFN-� (type II IFN) (AG129mice) have been successfully infected with DENV. The AG129model has been used to study dengue pathogenesis and to testantiviral compounds and therapeutic antibodies (6–9). However,the complete absence of IFN signaling renders these mice pro-foundly immunodeficient (reviewed in reference 5), which meansdata generated in these animals must be interpreted with caution.A recent report proposed an improvement to the AG129 model;mice lacking the IFN-�/� receptor but with intact IFN-� signalingwere highly susceptible to lethal infection with a mouse-adapteddengue virus strain and thus were proposed by the authors to bemore useful for vaccine testing than the AG129 mice (10). How-ever, the potential impact of a global lack of type I IFN receptor(IFNAR) signaling on the immune response to both dengue virusinfection and vaccination cannot be overlooked.
Studies using knockout mice have revealed that type I and IIIFNs play important but distinct roles in host defense againstDENV. As well as the AG129 mice (11), mice lacking signalingelements downstream of the IFN receptors (IFNRs) (STAT1/2), and STAT1 IFNAR double knockout mice are all highlysusceptible to DENV infection (12, 13). Mice deficient in justthe type I IFN receptor are also susceptible to high-titer DENVchallenge (14), while mice lacking the type II IFN receptoralone can resist even large doses of DENV without developingsignificant viremia or pathology (14, 15). Besides being impor-tant for the initial clearance of the virus, type I IFNs also en-hance B cell responses, Ig class switch (16–18), cell migration(19, 20), cross-presentation (21–24), CD4� T-cell activation(20, 25, 26), and cytotoxic T-lymphocyte (CTL) expansion(27–29). Therefore, any new mouse model for dengue shouldretain maximal interferon competence while achieving suffi-cient susceptibility to infection to be of practical use for immu-nological study of disease and vaccine screening.
In pursuit of this aim, we made use of a series of mice lackingthe type I IFNR on different subsets of immune cells and com-pared their responses to DENV2 infection and immunization witha candidate subunit vaccine. We demonstrated that deletion of thetype I IFN receptor on both LysM- and CD11c-expressing cells(predominantly macrophages and dendritic cells, respectively)renders mice susceptible to fatal DENV infection, whereas type IIFN signaling in CD4� cells is dispensable for the control ofDENV-2. Intriguingly, mice lacking the type I IFN receptor(IFNAR) on either LysM� or CD11c� cells were susceptible todisease but frequently recovered by 15 days postinfection (p.i.);furthermore, immunization of either of these mice with a denguevirus EDIII-capsid (EDIIIC-2) subunit vaccine (30, 31) inducedhigh titers of dengue-specific neutralizing antibodies. Therefore,we have developed a novel model which has revealed that type IIFN receptor signaling in CD11c- and LysM-expressing cells hasimportant and nonredundant roles in determining susceptibilityto dengue. Moreover, our minimally manipulative approach ren-ders this model highly appropriate for in vivo screening of DENVvaccine candidates, which could facilitate development of effec-tive prophylactic interventions for use in humans.
MATERIALS AND METHODSCells and virus. BHK-21 and C6/36 cells were purchased from the Amer-ican Type Culture Collection (http://www.atcc.org). U937 cells express-ing DC-SIGN were obtained by lentiviral transfection and subsequent cellsorting. All cells were maintained in minimal essential medium supple-mented with fetal bovine serum (5% to 10%).
For challenge experiments, dengue virus TSV01 or D2Y98P producedin C6/36 cells was used. For CD8� T-cell experiments, TSV01 virus wasused.
Mice. Female or male 6- to 8-week old IFN-�/�/� receptor-deficient(AG129) and wt Sv129 mice were purchased from B&K Universal Limitedwith permission from M. Aguet (ISREC, School of Life Sciences EcolePolytechnique Fédérale [EPFL]). LoxP-flanked ifnar1 (ifnar1fl/fl) (4) ani-mals were bred with mice that express Cre recombinase specifically in Tcells (CD4-Cre), macrophages, (LysM-Cre) (32), or CD11c� dendriticcells (CD11c-Cre) (33). All of these mice, including IFN-�/� receptor-deficient mice (IFNAR�/�), on a C57BL/6 background, were provided byUlrich Kalinke. All mice were bred and kept under specific-pathogen-freeconditions at the Biomedical Resource Centre, Singapore. For the gener-ation of bone marrow chimeras, recipient mice were irradiated with 950Gy from a Cs source. Twenty-four hours later, femurs and tibiae fromdonor mice were flushed with a syringe to isolate bone marrow cells. Atotal of 3 � 106 cells were transferred into irradiated recipient mice intra-venously. Mice were kept in individually ventilated cages and used be-tween 6 and 10 weeks of age. The mouse experiments were conductedaccording to the rules and guidelines of the Agri-Food and VeterinaryAuthority (AVA) and the National Advisory Committee for LaboratoryAnimal Research (NACLAR), Singapore. The experiments were reviewedand approved by the Institutional Review Board of the BiologicalResource Center, Singapore (IACUC protocols 100566, 120801, and060191).
Flow cytometry-based neutralization assay. The flow cytometry-based neutralization assay was performed as described previously (34).Briefly, 4G2 or heat-inactivated serum/plasma was serially diluted, and aconstant amount of virus was added. The antibody-virus mixture wasincubated at 37°C for 30 min and then added to U937-DC. After incuba-tion overnight, the infected cells were harvested, washed in phosphate-buffered saline (PBS), and fixed and permeabilized with Cytofix/Cy-toperm (BD). The percentage of infected cells was quantified by flowcytometry detecting intracellular E protein with Alexa Fluor 647-labeled4G2 antibody. Data were analyzed with GraphPad Prism software for thecalculation of the 50% neutralization titer (NT50).
Immunization and infection of mice. All mice were infected via theintraperitoneal (i.p.) route. AG129, Sv129, and bone marrow chimeraswere infected with 5 � 106 PFU of TSV01. IFNAR- and conditionalIFNAR knockout mice were infected with 1 � 107 PFU of D2Y98P (35,36). For survival experiments, the weights of the mice were monitoreddaily, and mice with loss of 20% of initial weight were considered mori-bund. EDIIIC-2 was expressed in Escherichia coli and purified as describedpreviously (31). Twenty micrograms of EDIIIC-2 or placebo (preparationcontaining synthetic oligonucleotide containing unmethylated CpG di-nucleotides [ODN] and alum only) was injected per immunization.
IgG ELISA. Ninety-six-well polystyrene plates were coated with con-centrated, UV-inactivated dengue virus. Plates were incubated overnightat 4°C. Before use, plates were washed three times in PBS (pH 7.2) con-taining 0.05% Tween 20 (PBS-T). Nonspecific binding was blocked with2% nonfat dry milk diluted in PBS (PBS-M) for 2 h at room temperature.After washing, sera were diluted 1:50 in PBS-M and heat inactivated for 1h at 55°C, and 3-fold serial dilutions were added to the wells. Plates wereincubated for 1 h at room temperature, followed by three washes withPBS-T. Peroxidase-conjugated rabbit anti-mouse IgG in PBS-M wasadded, followed by 1 h of incubation at room temperature and threeadditional washes with PBS-T. Tetramethylbenzidine (TMB) was used asthe enzyme substrate. The reaction was stopped with 1 M HCl, and theoptical densities were read at 450 nm using an automatic enzyme-linked
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immunosorbent assay (ELISA) plate reader. Endpoint titers were definedas the lowest dilution of plasma in which binding was 2-fold greater thanthe mean binding observed with the negative controls.
Determination of cytokines and virus in blood and organs. Virustiters in organs were determined from organs frozen in Trizol after weigh-ing and homogenization. Viral RNA was quantified by real-time quanti-tative reverse transcription-PCR (qRT-PCR) using primers and methodsreported previously (37). Virus titers in blood were determined by stan-dard plaque-forming assay using BHK-21 cells. The limit of detection was100 PFU/ml. Blood was collected in tubes containing sodium citrate, andplasma was subjected to alanine aminotransferase (ALT) measurement.Mouse tumor necrosis factor alpha (TNF-�), interleukin-6 (IL-6), IFN-�,IFN-�, IFN-�-induced protein 10 (IP-10) (eBioscence), and CXCL1 (KC/murine IL-8 homolog) (R&D Systems) protein levels in plasma were mea-sured by ELISA according to the manufacturers’ instructions.
T-cell restimulation. Mice were infected i.p. with 5 � 106 PFU ofTSV01. Five drops of blood were collected in 3 ml fluorescence-activatedcell sorter (FACS) buffer. Cells were collected by centrifugation, and redblood cells were lysed. Single-cell suspensions were stimulated with pep-tides (34) for 12 h, together with brefeldin for the last 5 h of stimulation,and cells were subsequently stained for intracellular IFN-� expression.
Statistical analysis. Statistical tests were performed with GraphPadPrism software, using Student’s t test, two-way analysis of variance(ANOVA), or chi-square tests as indicated in the figure legends.
RESULTSSusceptibility to DENV infection in conditional IFNAR knock-out mice. Dengue virus replicates efficiently in mice lacking type Iand II IFN receptors, but it is not known in detail which cell typesin these mice are the determinants of DENV susceptibility. Usingbone marrow chimeras, we first asked whether type I and II IFNRdeficiency on either hemopoietic or nonhemopoietic cells alonewas sufficient to enable viral replication. The chimeric mice wereinjected intraperitoneally (i.p.) with 5 � 106 PFU of the non-mouse-adapted DENV-2 strain TSV01, and blood samples weretaken daily for measurement of serum viremia (Fig. 1A). The lackof IFN receptors on nonhemopoietic cells did not lead to measur-able viral titers in serum, whereas mice lacking the IFN receptorson hemopoietic cells were effectively infected. Interestingly, whilethe positive-control mice (AG129¡AG129), which lacked IFNreceptors on all cells, remained highly viremic at day 6 postinfec-tion (p.i.), those animals deficient in IFN receptors on hemopoi-etic cells alone controlled their viremia and cleared the virus. Asthe majority of human infections with DENV cause a similarlylimited viremia, this led us to ask whether further refinements tothe specificity of IFN receptor deletion might enable developmentof an improved model of infection.
To further dissect the impact of type I IFN responsiveness inthe hemopoietic cell compartment on susceptibility to DENV in-fection, we generated a set of conditionally gene-targeted mice.Mice expressing a Cre recombinase under different immune cell-type-specific promoters were crossed with mice with a loxP-flanked IFNAR (IFNARfl/fl), generating mice lacking the type Ireceptor on CD4-expressing T cells (CD4-Cre�/� IFNARfl/fl), onLysM� cells, including macrophages, neutrophils, and some den-dritic cells (DCs) (LysM-Cre�/� IFNARfl/fl), on CD11c� conven-tional dendritic cells (cDCs) (CD11c-Cre�/� IFNARfl/fl), or onboth LysM- and CD11c-expressing cell populations (CD11c-Cre�/� LysM-cre�/� IFNARfl/fl). Because these mice retained typeII IFN receptors, they would not be susceptible to infection withthe DENV-2 strain used in the bone marrow chimera experiments(TSV01) and therefore were infected i.p. with 1 � 107 PFU of
FIG 1 Importance of type I IFN responsiveness on hematopoietic cellsfor control of dengue virus infection. (A) Bone marrow-chimeric mice(wt¡AG129, wt¡wt, AG129¡wt, and AG129¡AG129) were infected i.p.with 5 � 106 PFU dengue virus strain TSV01. Blood was drawn at indicatedtime points, and virus in serum was quantified by plaque assay. One represen-tative experiment of a total of two (with 3 to 5 mice per group) is shown. (B andC) Requirement for IFNAR expression on different leukocyte subsets. LysM-Cre�/� IFNARfl/fl, CD11c-Cre�/� IFNARfl/fl, CD11c-Cre�/� LysM-Cre�/�
IFNARfl/fl, CD4-Cre�/� IFNARfl/fl, wt, and IFNAR�/� mice were infected i.p.with 1 � 107 PFU of DENV strain D2Y98P. Mice were monitored daily, andmice with weight loss of more than 20% were considered moribund. Pooleddata from 5 experiments are shown, with totals of n � 21 for LysM-Cre�/�
IFNARfl/fl mice, n � 21 for CD11c-Cre�/� IFNARfl/fl mice, n � 10 for CD11c-Cre�/� LysM-Cre�/� IFNARfl/fl mice, n � 14 for CD4-Cre�/� IFNARfl/fl
mice, n � 13 for wt mice, and n � 19 for IFNAR�/� mice.
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DENV-2 D2Y98P (35, 36). The D2Y98P isolate harbors a muta-tion in the viral nonstructural protein NS4B, rendering the virusmore virulent in mice. Survival (Fig. 1B) and weight (Fig. 1C) ofmice were monitored daily over the 13 to 15 days following inoc-ulation. Deletion of the IFNAR on CD4� cells led to only a mini-mal loss of weight, and all mice survived the infection. In starkcontrast, all mice lacking the IFNAR on both LysM� and CD11c�
cells rapidly lost weight and died or had to be euthanized by day 5p.i. However, while the LysM-Cre�/� IFNARfl/fl and CD11c-Cre�/� IFNARfl/fl mice rapidly lost weight initially, and someeventually succumbed to the infection, by day 13 or 15, the sur-viving mice had recovered fully. This showed that the combinedaction of type I IFN signaling in LysM� and CD11c� cells is re-sponsible for prevention of clinical signs of early DENV infectionand that mice lacking the type I IFN receptor in just one of thesecell types are susceptible to DENV-2 but maintain sufficient im-mune competence to resolve the infection.
Dissemination of virus and cytokine production duringearly dengue virus infection of conditional IFNAR knockoutmice. To gain insight into the systemic effects of conditional dis-ruption of type I IFN signaling in immune cells, we infected miceas described above and sacrificed them on day 3 p.i. for measure-ment of viral titer in organs and serum. Mice lacking the IFNARon neutrophils and macrophages (LysM-Cre�/� IFNARfl/fl mice)contained significantly more DENV in their kidneys than CD11c-Cre�/� IFNARfl/fl mice, while viral titers in blood and other or-gans were similar (Fig. 2A to G). Surprisingly, the viral titers inblood and organs were also generally similar in groups of mice thatwould have been significantly more likely to recover from infec-tion (Fig. 1B) (LysM-Cre�/� IFNARfl/fl and CD11c-Cre�/�
IFNARfl/fl) as they were in groups that would likely have gone onto die by day 5 p.i. (CD11c-Cre�/� LysM-cre�/� IFNARfl/fl andIFNAR�/�). This shows that mice lacking IFNAR expression onCD11c- or LysM-expressing cells develop a full systemic viremiain the presence of an otherwise competent immune system, whichenables them to resolve infection rather than succumb to disease.
Thus far, we have shown that following DENV-2 inoculation,CD4-Cre�/� IFNARfl/fl mice have the highest survival rate, fol-lowed by CD11c-Cre�/� IFNARfl/fl, LysM-Cre�/� IFNARfl/fl, andthen CD11c-Cre�/� LysM-cre�/� IFNARfl/fl and IFNAR�/�
mice. In humans, the factors determining dengue disease severityare undefined, but cytokines are presumed to be important (38–41), and many studies have described increases in inflammatoryand regulatory cytokines during DENV infection (33). We there-fore asked whether there was any correlation between the levels ofspecific cytokines in sera at day 3 p.i. and the probability of sur-vival of infected mice. CD4-Cre�/� IFNARfl/fl and wt mice hadlow levels of all measured cytokines (Fig. 3A to F), as expectedfrom their low or undetectable viremia (Fig. 2A to G). Accord-ingly, groups of mice with higher viremia possessed higher overalllevels of cytokines in serum. The mice with the highest probabilityof survival, CD11c-Cre�/� IFNARfl/fl, had significantly higher se-rum levels of IL-6 and IFN-� than both LysM-Cre�/� IFNARfl/fl
and IFNAR�/� mice, suggesting that expression of these cytokinesmight be associated with survival. Interestingly, sustained IFN-�levels have been suggested to play a protective role in a humanchallenge model for dengue virus infection (42). On the otherhand, the IFNAR�/� mice, which would all have gone on to diefrom their infection, had significantly more TNF-� in their serathan CD11c-Cre�/� IFNARfl/fl mice, confirming previous find-
ings that higher levels of TNF-� are associated with disease sever-ity (41, 43). We also measured serum levels of the murine IL-8homologue CXCL1/KC, IFN-�, and IP-10. However, no signifi-cant differences between the conditional IFNAR and IFNAR�/�
mice were observed (Fig. 3D to F). Why double knockout mice didnot show the same cytokine profile as IFNAR�/� mice remains tobe further studied. The presence of viremia in mice correlatedwith elevated levels of alanine transaminase (ALT), which is indic-ative of liver damage (Fig. 3G). In summary, CD11c- and LysM-targeted IFNAR knockout mice developed full systemic viremiafollowing DENV-2 inoculation, which was accompanied by pro-lific cytokine production. Significantly higher levels of IFN-� andIL-6 and lower levels of TNF-� were evident in sera from CD11c-Cre�/� IFNARfl/fl mice and thus warrant further investigation aspotential correlates of protection.
Stronger CD8� T-cell responses to dengue virus infection inconditional IFNAR knockout mice compared to IFNAR�/�
mice. Type I IFNs are key drivers of T cell responses (22, 25).We therefore sought to test the ability of conditional IFNARmice to generate DENV-specific CD8� T cells. To this end, weinfected mice intraperitoneally with TSV01 and measuredIFN-�� CD8� T cells in the blood at days 3, 6, and 8 postin-fection. Surprisingly, IFNAR�/� mice showed minimal expan-sion of DENV-specific CD8� T cells. In contrast, wild-typemice and CD11c-Cre�/� IFNARfl/fl and LysM-Cre�/�IFNARfl/fl
mice responded with a swift increase of virus-specific CD8� Tcells, peaking at day 6 p.i. (Fig. 4). Of note, the peak of the CD8�
T-cell response correlated with the gain of weight and recovery ofsurviving mice (Fig. 1C), highlighting the importance of CD8� Tcells for viral clearance.
Immune responses of conditional IFNAR knockout mice to adengue subunit vaccine candidate. While mice lacking theIFNAR on all cells have been used as models of dengue virus in-fection, the fact that type I IFNs are essential drivers of B- andT-cell responses (18, 22, 25, 40) renders these mice of questionableuse in the assessment of potential vaccine candidates. We there-fore asked whether our immunocompetent mice with conditionaldeletion of the IFNAR in either CD11c- or LysM-expressing cellswould be able to generate more effective B-cell responses aftervaccination with a recombinant EDIIIC-2 subunit vaccine candi-date (31). Groups of mice were immunized subcutaneously threetimes, each separated by 2 weeks, with 20 g of EDIIIC-2 vaccine.All mice generated high levels of DENV-2 virion-specific antibod-ies as measured by ELISA (Fig. 5A) and also showed comparableneutralizing titers (Fig. 5B). Surprisingly, however, CD11c-Cre�/�IFNARfl/fl mice were best protected against subsequent vi-ral homologous challenge with DENV-2 strain D2Y98P (Fig.5C) and showed the highest survival rate, followed by LysM-Cre�/�IFNARfl/fl mice (Fig. 5D). To compare the neutralizingeffect of antibodies produced by wt mice with those producedby conditional knockout mice, we immunized mice withEDIIIC-2 as described above and transferred 100 l of seruminto dengue virus-susceptible AG129 mice. All mice suc-cumbed to infection within 4 to 5 days postinfection. While thedifference in viral titers was not statistically significant, anti-bodies produced by the most susceptible mice (IFNAR�/�)induced the highest antibody-dependent enhancement (ADE)when transferred into wt mice, suggesting a lower protectivecapacity of the antibodies produced in IFNAR�/� mice com-pared to those produced in wt and conditional IFNAR mice
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(data not shown). Similar results have been published by others(44) with the conclusion that antibodies alone can cause ADE,while the combination of antibody and T-cell response seemskey for protection. This highlights once again the urgent needfor better, immunocompetent mouse models that are able to
generate functional antibody and T-cell responses and demon-strates that the conditional IFNAR knockout mice not only aresuitable models to study DENV infection biology but also aresufficiently immunocompetent to raise effective antibody andT-cell responses to an adjuvanted subunit vaccine.
FIG 2 Virus loads in organs and blood of mice infected with D2Y98P. Mice were inoculated i.p. with 1 � 107 PFU of D2Y98P, and organs were harvested 3 dayspostinfection. Viral loads were determined by real-time PCR in spleen (A), liver (B), lung (C), brain (D), kidney (E), and inguinal lymph nodes (LN) (F) and byplaque assay in serum (G). Each symbol represents one mouse. Data are presented as means standard deviations (SD). Statistical analysis of differences betweenLysM-Cre�/� IFNARfl/fl and CD11c-Cre�/� IFNARfl/fl, CD11c-Cre�/� IFNARfl/fl and IFNAR�/�, and LysM-Cre�/� IFNARfl/fl and IFNAR�/� mice wasperformed using Student’s t test. ***, P � 0.001; **, P � 0.01; *, P � 0.05. Nonsignificant differences are not indicated.
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FIG 3 Systemic levels of soluble mediators in infected mice. Mice were infected i.p. with 1 � 107 PFU of D2Y98P, and sera were harvested 3 days postinfection.Levels of IL-6 (A), IFN-� (B), TNF-� (C), IP-10 (D), CXCL1/KC (E), and IFN-� (F) were determined by ELISA. Alanine transaminase (ALT) levels (G) werequantified using the Cobas C111 chemistry analyzer (Roche). Each symbol represents one mouse. Data are presented as means SD. Statistical analysis ofdifferences between LysM-Cre�/� IFNARfl/fl and CD11c-Cre�/� IFNARfl/fl, LysM-Cre�/� IFNARfl/fl and IFNAR�/�, and CD11c-Cre�/� IFNARfl/fl andIFNAR�/� mice was performed using one-way ANOVA with Bonferroni’s posttest. ****, P � 0.0001; **, P � 0.01; *, P � 0.05. Nonsignificant differences are notindicated.
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DISCUSSION
Despite huge efforts over the last 70 years and a growing clinicalneed, there remains no treatment or vaccine for dengue. Denguevaccine research has faced numerous challenges, including uncer-tainties on immune correlates of protection and enhancementand the lack of suitable animal models in which to dissect diseasepathways and test novel therapeutics or vaccines. The first animalmodels established were immunocompetent mice in which a hightiter of DENV was delivered intracranially (45, 46). This methodcaused neurotropic disease and paralysis, neither of which is typ-ically observed in humans. More recently, immunocompromisedmice lacking type I and/or type II IFN receptors have been gener-ated (10, 11). Dengue infection in these mice recapitulates someaspects of the human disease, such as cytokine storm, vascularleakage, TNF-� production, hemorrhage, and nonparalytic death(36). However, valid concerns have been raised regarding the ex-trapolation of data generated in these mice to the human setting.In fact, it is known that type I IFN receptor triggering is requiredfor maximal T-cell expansion (47), cross-priming of CD8� T cells(22), and promotion of clonal expansion and memory T-cell gen-eration (29); additionally, type I IFNs directly stimulate naive Tcells (48, 49) and enhance B-cell responses (18, 25, 50, 51). Thus,mice defective in these pathways are unlikely to recapitulate manyfeatures of a natural immune response to dengue virus infection.
During viral infections, macrophages and cDCs play an impor-tant role in both early innate immunity and in instructing thedevelopment of the adaptive immune response. Our data demon-strate that protection of mice from DENV-2 infection depends onIFN signaling in LysM-expressing cells, including macrophagesand neutrophils and CD11c-expressing cDCs. Mice lacking the
IFNAR on either LysM� or CD11c� cells were susceptible toDENV-2 but frequently began to recover from day 5 p.i. As theCD8� T-cell response to DENV also begins around this time (52),this leads us to speculate that these cells may be important inresolving DENV-2 infection in conditional IFNAR knockoutmice. In line with this, conditional IFNAR knockout mice showedhigher DENV-specific CD8� responses than IFNAR�/� mice,which succumbed to infection. Further investigation will reveal towhat extent CD8�and CD4� T cells are necessary for recovery andhow type I IFNs are involved in this process.
Our immunization studies using an alum- and ODN-adju-vanted EDIIIC-2 subunit vaccine highlighted the importance ofIFN signaling in macrophages for the development of an effectiveadaptive immune response. A commonly used readout of vaccineefficacy is serum titer of dengue-specific and neutralizing antibod-ies. Our data demonstrate that neutralizing antibody titers aloneare of limited value in predicting the ability of a vaccine to protectfrom viral challenge in mice, which is consistent with previousfindings in humans (53, 54). Although all mouse strains testedproduced comparable amounts of DENV-2-specific IgG and sim-ilar titers of neutralizing antibodies, CD11c-Cre�/� IFNARfl/fl
mice were significantly more protected against challenge. Unex-pectedly, although viral titers in serum of untreated and placebo-treated CD11c-Cre�/� IFNARfl/fl mice at day 3 p.i. were compa-rable (Fig. 2G and 5C), the placebo control group succumbed tothe infection (Fig. 5D), whereas untreated mice mostly survived(Fig. 1B). One reason for this unexpected result might be that theadjuvant in the placebo activated immune cells and rendered themice more susceptible to immunopathology-related disease out-come. Not only does this reemphasize the finding that peak virus
FIG 4 T-cell response of mice infected with DENV. Mice were infected with 5 � 106 PFU dengue virus strain TSV01, and blood was drawn on the indicated dayspostinfection. Cells were collected by centrifugation and restimulated with peptides for the quantification of IFN-� production in CD8� cells. (A) IntracellularIFN-� was measured in blood CD8� cells of infected LysM-Cre�/� IFNARfl/fl, CD11c-Cre�/� IFNARfl/fl, IFNAR�/�, and wt mice. Representative FACS graphsgated on CD8� lymphocytes for each group at day 6 postinfection are shown. (B) Quantitative analysis of IFN-� production. Bars are means standard errorsof the means (SEM) from two independent experiments with a total of 4 to 8 mice.
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titers are not necessarily predictive of final disease outcome duringDENV infection of mice (Fig. 1 and 5) but highlights the fact thatthe delicate immunological balance determining recovery versusdeath from dengue depends on currently unidentified factorswhich are likely linked to IFNAR expression on CD11c� and/orLysM� cells. Taken together, our data reveal that the conditionalIFNAR knockout mice are appropriate models for the study ofdengue virus immunology and pathology. They have begun toprovide insight into the association of peak viremia, cytokine pro-duction, and the role of type I IFNs in determining disease severityand outcome, and further study may advance the search for theelusive immune correlates of protection. Moreover, CD11c-Cre�/� IFNARfl/fl mice are a more relevant model than eitherAG129 or global IFNAR knockout models with which to screendengue vaccine candidates. The development and study of theseconditional IFNAR knockout mice should facilitate progress to-ward the development of effective dengue treatments and vaccinesfor clinical use.
ACKNOWLEDGMENTS
We thank Lucy Robinson of Insight Editing London for greatly improvingour manuscript.
This work was supported by the Agency for Science, Technology andResearch (A*STAR), Singapore.
The Institute for Experimental Infection Research, Twincore, Centerfor Experimental and Clinical Infection Research, is a joint venture be-tween the Helmholtz Centre for Infection Research and the HannoverMedical School, Hannover, Germany.
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